Enzymes catalyze an impressively diverse set of biologically-essential chemical reactions with huge accelerations in rate over the uncatalyzed processes. The fundamental understanding of how enzymes achieve these enormous rate accelerations has been the focus of much biochemically-related research over the past decades. Much interest has been placed on obtaining descriptive static structures of enzymes at atomic resolution. Although these models provide exceptional details in relation to the lowest energy conformer(s), the important dynamical information regarding enzymes is lost. A link between protein dynamics and the chemical step in enzyme catalysis was initiated with the discovery of enzymatic hydrogen tunneling reactions. Quantum hydrogen tunneling comprises an important niche of chemical catalysis in biology. Soybean lipoxygnease (SLO) is a member of the lipoxygenases that oxidize long-chain fatty acids and have been implicated in numerous inflammation-related diseases. More importantly, SLO exhibits the benchmark kinetic parameters for nuclear tunneling, and thus, has emerged as the prototypical model system for enzymatic nuclear tunneling. Hydrogen tunneling requires heavy atom motions, in both the substrate and protein matrix, to properly adjust the necessary energetics and donor-acceptor (DA) distances for effective hydrogenic wave function overlap. Theoretical treatments have suggested that the donor and acceptor are compressed in nuclear tunneling to a distance less than van der Waals radii, though the origin of this compressed DA distance is yet unresolved. These putative short distances are hypothesized to be achieved from either active site compaction or the dynamic sampling of active protein conformers. Together, these geometric and dynamic factors may account for the huge rate accelerations in enzymatic hydrogen tunneling reactions. This proposal outlines two main goals to address these very important, yet challenging, fundamental problems pertaining to the function of enzymes. These goals will be accomplished through two Specific Aims. First, the ground-state donor-acceptor distances, between the catalytic metal in SLO and isotopically-labeled substrates (linoleic acid), will be measured with ENDOR spectroscopy. Using established synthetic approaches, the Applicant will synthesize specific 13C labeled linoleic acid. ENDOR experiments conducted for a series of SLO variants with these labeled substrates will allow for a direct experimental measure of the DA distance, substrate orientation, and flexibility at the active site. Second, the impact of mutation and temperature on protein flexibility, both proximal and distal to the active site, will e probed using hydrogen-deuterium exchange mass spectrometry (HDX-MS). Comparison of the results for the wild type SLO to catalytically- impaired mutants will provide insight into catalytically-relevant protein motions. Furthermore, this study will underscore the structural interplay between local DA distance sampling and more global protein motions, which are key aspects of the accelerated rate enhancements in biological hydrogen tunneling reactions.